The selective and ordered adhesion and colonization of commensal bacteria on the teeth can be explained at least in part by the expression of discrete binding sites on the coating salivary film or pellicle. Since the molecular organization of the salivary molecules in the pellicle is unknown, this project tested the hypothesis that specific adhesive binding epitopes would be identified by reaction with anti-idiotypical (anti-ids) monoclonal antibodies (MAbs2) raised against anti-Streptococcus sanguis adhesin monoclonal antibodies (MAbs1). Several MAbs2 were developed and used to probe the surface of saliva-coated hydroxylapatite (sHA), an in vitro model of the enamel pellicle. The sHA bound anti-id MAbs2 to inhibit adhesion of an adhering strain of S. sanguis. These anti-ids reacted with a complex of the light chain of secretory IgA and alpha-amylase and the individual proteins. These proteins contain epitopes that serve as binding sites for adhesins of an adherent strain of S. sanguis. Additional binding epitope(s) for other strains and species may also exist on sHA. To continue to define the molecular organization of the pellicle, this project proposes to characterize the binding sites for S. sanguis. The number, affinity and spatial organization of these binding sites are hypothesized to specify the order of preferred adhesion of the early flora on the teeth. Specifically, to learn if these binding sites could be used generally by early adhering species, (1) a library of strains of S. sanguis (IgA protease+, amylase binding) and other selected species will be compared for reaction with the existing MAbs1 and inhibition of binding to sHA by the corresponding MAbs2. Some species, such as S. gordonii, may bind to other sites containing proline-rich proteins (PRPs), for example. To identify binding site epitopes on sHA that contain PRPs, (2) new MAbs1 and their MAbs2 (anti-ids) will be developed. To model how salivary proteins may act on pellicle to form specific binding site(s0 for early colonizers, (3) the structure and topological distribution of sIgA light chain, amylase, and the sIgA-amylase complex will be characterized. The access to their respective binding sites on pellicle may influence the competition between S. sanguis and S. gordonii for adhesion. The specific binding sites on sHA for S. sanguis and S. gordonii will be characterized (4) by competitive inhibition assays with specific MAbs2 Fab fragments and their topological proximity determined at molecular dimensions. To ensure that the reactions with sHA in vitro accurately model the dynamics in the oral cavity, (5) enamel chips will be coated with saliva of volunteers ex vivo, incubated with mAbs2, placed in the mouths of volunteers, and changes in the adhesion and colonization of early flora will be determined. These studies will provide the first definitive demonstration that molecular organization of the salivary pellicle confers binding specificity for S. sanguis and other early streptococcal colonizers of teeth.